The identification of borders of cell bodies and/or cell nuclei in a microscopy image of a sample may be referred to as segmentation of cell bodes and/or nuclei in the image. To image structures of cells in a sample (e.g., for segmentation), staining or labeling techniques are often used to enhance contrast between different types of cell structures. For example, a specimen may be stained with dyes that react with DNA or RNA (e.g., ethidium bromide), or dyes that interact differently with the nucleus and the cytoplasm of a cell (e.g., hematoxylin-eosin). As another example, labels, which may be one or more colored agents (chromopores) and/or or one or more fluorescent agents (fluorophores), are useful in identifying a desired substance in a cell structure based on the presence of a specific tag. Such staining and labeling techniques aid in the identification of cell structures; however, staining or labeling using external contrast agents may affect the structure or other properties of interest of the sample being imaged. Further, in general, such staining or labeling cannot be performed on live cells.
Conventional phase-contrast imaging techniques (e.g., Zernike, different interference contrast (DIC)) enable imaging of cell monolayers with improved contrast as compared with bright-field imaging. These phase-contrast techniques, which do not require staining or labeling, generally can provide sufficient image contrast, but at the expense of additional optical components, light sources and alignment procedures.
Conventional phase imaging can be approximated as processing a series of traditional transmitted light images (e.g., by subtraction of in and out of focus images). While providing an image with enhanced edge features, the contrast is often not sufficient to insure reliable automated segmentation of cells and/or nuclei.
Further, neither conventional optical imaging with labels and stains, nor conventional phase-contrast imaging provides quantitative thickness information regarding cell monolayers.